A new approach to the isolation of common antigen (CA) of the Enterobacteriaceae has been employed. The method involves procedures designed to separate subcellular fractions and preserve intact cell structures containing common antigen. It has resulted in the observation of phenomena associated with the common antigenic response which are indeed different from what has been previously reported. Two particulate fractions (one predominantly cell envelope and the other cell envelope fragments and vesicular membranous components), and a soluble fraction have been isolated from Escherichia coli 014, Salmonella typhimurium. Klebsiella pneumoniae, Salmonella minnesota smooth 218 and derived rough mutants Ra60, rb345, rc5, rd7, rd24, and re595. All cell fractions of all percent organisms except the soluble fraction of Klebsiella pneumoniae were immunogenic for CA and O in rabbits. Antigen associated immunosuppressive was not observed in any fractions. The CA antigenic determinant (both Indirect Hemagglutination and Hemagglutination Inhibition reactivities) were present in all fractions of parent organisms. All fractions of rough mutants Ra and Rb were immunogenic and also exhibited serologic antigenic activity for CA, but Rc, Rd1, Rd2, and Re were not. The cell envelop does not appear to be the exclusive locus of CA since immunological activity was observed with all fractions. Binding of the two particulate fractions of RBC's made the O antigenic determinant (which is clearly present in these fractions from hemagglutination inhibition determinations) unavailable for reaction with its specific antibody, while allowing a reaction between the CA determinant and its antibody. Results obtained with rough mutants suggest that the O-antigenic side chains and the terminal N-acetylglucosamine of the core polysaccharide are not involved in the antigenic determinant of CA.